This invention relates generally to agricultural biotechnology, and more specifically to methods and genetic materials for conferring resistance to fungi and/or viruses in plants.
The subject of plant protection against pathogens remains the area of utmost importance in agriculture. Many commercially valuable agricultural crops are prone to infection by plant viruses and fungi capable of inflicting significant damage to a crop in a given season, and drastically reducing its economic value. The reduction in economic value to the farmer in turn results in a higher cost of goods to ultimate purchasers. Several published studies have been directed to the expression of plant virus capsid proteins in a plant in an effort to confer resistance to viruses. See, e.g., Abel et al., Science 232:738-743 (1986); Cuozzo et al., Bio/Technology 6:549-557 (1988); Hemenway et al., EMBO J. 7:1273-1280 (1988); Stark et al., Bio/Technology 7:1257-1262 (1989); and Lawson et al., Bio/Technology 8:127-134 (1990). However, the transgenic plants exhibited resistance only to the homologous virus and related viruses, but not to unrelated viruses. Kawchuk et al., Mol. Plant-Microbe Interactions 3(5):301-307 (1990), disclose the expression of wild-type potato leaf roll virus (PLRV) coat protein gene in potato plants. Even though the infected plants exhibited resistance to PLRV, all of the transgenic plants that were inoculated with PLRV became infected with the virus and thus disadvantageously allowed for the continued transmission of the virus such that high levels of resistance could not be expected. See U.S. Pat. No. 5,304,730.
Lodge et al., Proc. Natl. Acad. Sci. USA 90:7089-7093 (1993), report the Agrobacterium tumefaciens-mediated transformation of tobacco with a cDNA encoding wild-type pokeweed antiviral protein (PAP) and the resistance of the transgenic tobacco plants to unrelated viruses. PAP, a Type I ribosome-inhibiting protein (RIP) found in the cell walls of Phytolacca americana (pokeweed), is a single polypeptide chain that catalytically removes a specific adenine residue from a highly conserved stem-loop structure in the 28S rRNA of eukaryotic ribosomes, and interferes with elongation factor-2 binding and blocking cellular protein synthesis. See, e.g., Irvin et al., Pharmac. Ther. 55:279-302 (1992); Endo et al., Biophys. Res. Comm., 150:1032-36 (1988); and Hartley et al., FEBS Lett. 290:65-68 (1991). The observations by Lodge were in sharp contrast to previous studies, supra, which reported that transgenic plants expressing a viral gene were resistant to that virus and closely related viruses only. See also Beachy et al., Ann. Rev. Phytopathol. 28:451-74 (1990); and Golemboski et al., Proc. Natl. Acad. Sci. USA 87:6311-15 (1990). Lodge also reports, however, that the PAP-expressing tobacco plants (i.e., above 10 ng/mg protein) tended to have a stunted, mottled phenotype, and that other transgenic tobacco plants that accumulated the highest levels of PAP were sterile. RIPs have proven unpredictable in other respects such as target specificity. Unlike PAP which (as demonstrated in Lodge), ricin isolated from castor bean seed is 1000 times more active on mammalian ribosomes than plant ribosomes. See, e.g., Harley et al., Proc. Natl. Acad. Sci. USA 79:5935-5938 (1982). Barley endosperm RIP also shows very little activity against plant ribosomes. See, e.g., Endo et al., Biochem. Biophys. Acta 994:224-226 (1988) and Taylor et al., Plant J. 5:827-835 (1984).
Fungal pathogens contribute significantly to the most severe pathogen outbreaks in plants. Plants have developed a natural defense system, including morphological modifications in their cell walls, and synthesis of various anti-pathogenic compounds. See, e.g., Boller et al., Plant Physiol 74:442-444 (1984); Bowles, Annu. Rev. Biochem. 59:873-907 (1990); Joosten et al., Plant Physiol. 89:945-951 (1989); Legrand et al., Proc. Natl. Acad. Sci. USA 84:6750-6754 (1987); and Roby et al., Plant Cell 2:999-1007 (1990). Several pathogenesis-related (PR) proteins have been shown to have anti-fungal properties and are induced following pathogen infection. These are different forms of hydrolytic enzymes, such as chitinases and xcex2-1,3-glucanases that inhibit fungal growth in vitro by destroying fungal cell walls. See, e.g., Boller et al., supra; Grenier et al., Plant Physiol. 103:1277-123 (1993); Leah et al., J. Biol. Chem. 266:1464-1573 (1991); Mauch et al., Plant Physiol. 87:325-333 (1988); and Sela-Buurlage Buurlage et al., Plant Physiol. 101:857-863 (1993).
Several attempts have been made to enhance the pathogen resistance of plants via recombinant methodologies using genes encoding pathogenesis-related proteins (such as chitinases and xcex2-1,3-glucanases) with distinct lytic activities against fungal cell walls. See, e.g., Broglie et al., Science 254:1194-1197 (1991); Vierheilig et al., Mol. Plant-Microbe Interact. 6:261-264 (1993); and Zhu et al., Bio/Technology 12:807-812 (1994). Recently, two other classes of genes have been shown to have potential in conferring disease resistance in plants. Wu et al., Plant Cell 7:1357-1368 (1995), report that transgenic potato expressing the Aspergillus niger glucose oxidase gene exhibited increased resistance to Erwinia carotovora and Phytophthora infestans. The hypothesis is that the glucose oxidase-catalyzed oxidation of glucose produces hydrogen peroxide, which when accumulates in plant tissues, leads to the accumulation of active oxygen species, which in turn, triggers production of various anti-pathogen and anti-fungal mechanisms such as phytoalexins (see Apostol et al., Plant Physiol. 90:109-116 (1989) and Degousee, Plant Physiol. 104:945-952 (1994)), pathogenesis-related proteins (Klessig et al., Plant Mol. Biol. 26:1439-1458 (1994)), strengthening of the plant cell wall (Brisson et al., Plant Cell 6:1703-1712 (1994)), induction of systemic acquired resistance by salicylic acid (Chen et al., Science 162:1883-1886 (1993)), and hypersensitive defense response (Levine et al., Cell 79:583-593 (1994)).
In addition to the studies on virus resistance in plants, RIPs have been studied in conjunction with fungal resistance. For example, Logeman et al., Bio/Technology 10:305-308 (1992), report that a RIP isolated from barley endosperm provided protection against fungal infection to transgenic tobacco plants. The combination of barley endosperm RIP and barley class-II chitinase has provided synergistic enhancement of resistance to Rhizoctonia solani in tobacco, both in vitro and in vivo. See, e.g., Lea et al., supra; Mauch et al., supra; Zhu et al., supra; and Jach et al., The Plant Journal 8:97-109 (1995). PAP, however, has not shown antifungal activity in vitro. See Chen et al., Plant Pathol. 40:612-620 (1991), which reports that PAP has no effect on the growth of the fungi Phytophthora infestans, Colletotrichum coccodes, fusarium solani, fusarium sulphureum, Phoma foreata and Rhizoctonia solani in vitro.
Hence, a need remains for a means by which to confer broad spectrum virus and/or fungus resistance to plants without causing cell death or sterility, and which requires a minimum number of transgenes.
The present invention is directed to PAP mutants having reduced phytotoxicity, and which exhibit PAP biological activity in plants. By xe2x80x9cPAP biological activity,xe2x80x9d it is meant PAP anti-viral activity and/or PAP anti-fungal activity. One preferred group of PAP mutants is characterized by at least one amino acid substitution in the N-terminus of mature PAP, such as a substitution for the Glycine 75 residue or the Glutamic acid 97 residue. Another group of PAP mutants is characterized by a truncation of as many as 38 amino acids at the N-terminus of mature PAP. Yet another preferred group of PAP mutants is characterized by mutations such as truncations in the C-terminal region of mature PAP. More preferred are PAP mutants truncated at their C-terminus by at least about 26 to about 76 mature PAP amino acids (not counting the 29-amino acid C-terminal extension of wild-type PAP). A further group of PAP mutants are enzymatically inactive and do not exhibit PAP anti-viral activity in vitro or in planta; yet, they exhibit PAP anti-fungal activity in plants. The PAP mutants of the present invention may also include the 22-amino acid N-terminal signal sequence and/or the C-terminal extension of wild-type PAP.
The present invention also provides DNA molecules encoding the PAP mutants, which may or may not also encode the 22-amino acid N-terminal signal sequence of mature PAP and/or the 29-amino acid C-terminal extension of wild-type PAP. The DNAs can be operably linked to a promoter functional in procaryotic cells (e.g., E. coli), or eukaryotic cells such as plants, and then stably transformed into a vector functional in said cells. Hosts, e.g., procaryotic or eukaryotic cells (e.g., yeast or plants), stably transformed with a mutant PAP-encoding DNA are also provided, as well as protoplasts stably transformed with the DNAs. Transgenic plants and seed containing the DNAs are also provided. Expression of the DNAs in the transgenic plants confers broad spectrum virus and/or fungus resistance upon the plants without being as phytotoxic to the plant as wild-type PAP. Plants included within the scope of the present invention are monocots, such as cereal crops, and dicot plants.
The present invention further provides a method for identifying a PAP mutant having reduced phytotoxicity and which exhibits PAP biological activity in plants. The method involves the steps of providing a transformed eukaryotic cell such as yeast containing a mature PAP-encoding DNA molecule operably linked to an inducible promoter functional in the eukaryotic cell. The PAP-encoding DNA is mutagenized prior to transformation, or the transformed cell is mutagenized (i.e., the mutagenesis is performed after the cell is transformed with the PAP construct). The thus-transformed cells are cultured in a suitable medium, and after a predetermined time, an inducer is added to the medium to cause expression of the DNA molecule. A determination is then made as to whether the survival of cultured cells is due to the expression of a mutant PAP. Such mutant PAPs which exhibit a substantial lack of toxicity to the host would be considered as PAP mutants which exhibit reduced phytotoxicity. The thus-identified PAP mutants which also exhibit broad spectrum virus and/or fungus resistance, as determined by in vitro (e.g., by exogenous application of the virus or fungus), or in vivo (e.g., by expression in transgenic plants); would also be considered as PAP mutants which retain PAP biological activity in plants. The present invention further provides isolated and purified PAP mutants identified by the aforesaid method.
Transgenic plants expressing DNAs encoding the PAP mutants of the present invention exhibit reduced phytotoxicity compared to transgenic plants that produce mature, wild-type PAP, (xe2x80x9cPAPxe2x80x9d), or variant PAP, i.e. PAP-v, but also exhibit anti-viral and/or anti-fungal activities. By the term xe2x80x9creduced phytotoxicity,xe2x80x9d it is meant that a transgenic plant which expresses a mutant PAP-encoding DNA exhibits a normal and fertile phenotype and does not exhibit the stunted, mottled phenotype characteristic of transgenic plants that produce mature PAP (as disclosed in Lodge for example). By xe2x80x9cwild-type PAP,xe2x80x9d it is meant the PAP amino acid sequence 1-262, the 22-amino acid N-terminal signal peptide (xe2x80x9cthe N-terminal signal sequence of wild-type PAPxe2x80x9d), and the 29 amino acid C-terminal extension (amino acids enumerated 263-291), all illustrated in Table I below as SEQ ID NO:2. The corresponding nucleotide sequence is set forth as SEQ ID NO:1. Thus, by the terms xe2x80x9cwild-type, mature PAP,xe2x80x9d or xe2x80x9cmature PAPxe2x80x9d, it is meant the PAP amino acid sequence 1-262 shown in Table I.
Table I further shows PAP-v amino acids and corresponding nucleotides in proper alignment with wild-type PAP. Basically, the amino acid sequence of PAP-v differs from that of wild-type PAP in terms of a Leu20Arg (i.e., an arginine residue at position 20 of mature PAP as opposed to a leucine residue) and a Tyr49His substitution. The third change in the PAP-v nucleotide sequence (TCGxe2x86x92TCA codon for the first occurring Ser in the signal sequence) has no effect on the amino acid sequence. Table I also shows 5xe2x80x2 and 3xe2x80x2 non-coding, flanking sequences. Upon expression in eukaryotic cells, the N-terminal 22-amino acid sequence of wild-type PAP is co-translationally cleaved, yielding a polypeptide having a molecular weight of about 32 kD, which is then further processed by the cleavage of the C-terminal 29-amino acids (xe2x80x9cthe C-terminal extension of wild-type PAPxe2x80x9d or xe2x80x9cPAP (263-292)xe2x80x9d), yielding mature, wild-type PAP (hereinafter xe2x80x9cPAP (1-262)xe2x80x9d) (i.e., that which is isolated from Phytolacca americana leaves), having a molecular weight of about 29 kD. See Irvin et al., Pharmac. Ther. 55:279-302 (1992); Dore et al., Nuc. Acids Res. 21(18):4200-4205 (1993); Monzingo et al., J. Mol. Biol. 233:705-15 (1993); Turner et al., Proc. Natl. Acad. Sci. USA 92:8448-8452 (1995).
By the phrase xe2x80x9cPAP anti-viral activity,xe2x80x9d it is meant that the expression of a mutant PAP of the present invention in a transgenic plant confers broad spectrum virus resistance, i.e., resistance to or the capability of suppressing infection by a number of unrelated viruses, including but not limited to RNA viruses e.g., potexviruses such as (PVX, potato virus X), potyvirus (PVY), cucumber mosaic virus (CMV), tobacco mosaic viruses (TMV), barley yellow dwarf virus (BYDV), wheat streak mosaic virus, potato leaf roll virus (PLRV), plumpox virus, watermelon mosaic virus, zucchini yellow mosaic virus, papaya ringspot virus, beet western yellow virus, soybean dwarf virus, carrot read leaf virus and DNA plant viruses such as tomato yellow leaf curl virus. See also Lodge, Tomlinson et al., J. Gen. Virol. 22:225-232 (1974); and Chen et al., Plant Pathol. 40:612-620 (1991).
By the phrase xe2x80x9cPAP anti-fungal activityxe2x80x9d, it is meant that the mutant PAPs of the present invention confer broad spectrum fungal resistance to plants. The mutant PAPs of the present invention provide increased resistance to diseases caused by plant fungi, including those caused by Pythium (one of the causes of seed rot, seedling damping off and root rot), Phytophthora (the cause of late blight of potato and of root rots, and blights of many other plants), Bremia, Peronospora, Plasmopara, Pseudoperonospora and Sclerospora (causing downy mildews), Erysiphe graminis (causing powdery mildew of cereals and grasses), Verticillium (causing vascular wilts of vegetables, flowers, crop plants and trees), Rhizoctonia (causing damping off disease of many plants and brown patch disease of turfgrasses), Fusarium (causing root rot of bean, dry rot of potatoes), Cochliobolus (causing root and foot rot, and also blight of cereals and grasses), Giberella (causing seedling blight and foot or stalk rot of corn and small grains), Gaeumannomyces (causing the take-all and whiteheads disease of cereals), Schlerotinia (causing crown rots and blights of flowers and vegetables and dollar spot disease of turfgrasses), Puccinia (causing the stem rust of wheat and other small grains), Ustilago (causing corn smut), Magnaporthae (causing summer patch of turfgrasses), and Schlerotium (causing southern blight of turfgrasses). Other important fungal diseases include those caused by Cercospora, Septoria, Mycosphoerella, Glomerella, Colletotrichum, Helminthosporium, Alterneria, Botrytis, Cladosporium, and Aspergillus.
Applicant also believes that the mutant PAPs of the present invention confer increased resistance to insects, bacteria and nematodes in plants. Important bacterial diseases include those caused by Pseudomonas, Xanthomonas, Erwinia, Clavibacter and Streptomyces.
The PAP mutants of the present invention differ from wild-type PAP substantially as follows: (1) those which exhibit altered compartmentalization in vivo; (2) C-terminal mutants, including but not limited to deletion or frameshift mutants; (3) N-terminal mutants; and (4) active-site mutants. The first category of PAP mutants may have altered compartmentalization properties in vivo; that is, they may not be localized in the same subcellular compartment as wild-type PAP. While not intending to be bound to any particular theory of operation, Applicant believes that these PAP mutants are unable to undergo co-translational processing (to remove the 22 amino acid signal peptide) and/or post-translational processing (to remove the 29-amino acid C-terminal fragment) in yeast, which results in substantially diminished or negligible cytotoxicity. These mutants are also non-phytotoxic. What is particularly surprising or unexpected about the function of these mutant PAPs in vivo is that the mutations are located within the sequence encoding the mature PAP (1-262), and not within the N-terminal signal peptide or the 29-amino acid C-terminal extension. In addition, the mutant PAPs are enzymatically active in inhibiting translation in vitro, indicating that phytotoxicity is not solely a function of enzymatic activity. Preferred PAP mutants include a conservative point mutation such that wild-type PAP amino acid residue 75 glycine (Gly75) is changed to valine, alanine, isoleucine or leucine, or (2) a conservative or non-conservative point mutation at wild-type PAP amino acid residue 97 Glutamic acid (Glu97). More preferred PAP mutants are PAP (1-262, Gly75Val) and PAP (1-262, Glu97Lys), the respective DNAs of which can be prepared simply by changing the wild-type GGT codon for glycine75 to GTT (valine), and the GAA codon for glutamic acid 97 to AAA (lysine). Other PAP mutants having altered compartmentalization properties can be identified by the selection method described below. Dore et al., supra, disclose an Arg67Gly PAP mutant (numbered in Dore as Arg 68Gly due to the presence of an N-terminal methionine residue), but which is toxic to eukaryotic cells and non-toxic to procaryotic cells such as E. coli. This mutant is not included within the scope of the present invention.
The second category of PAP mutants of the present invention have deletions or amino acid substitutions in the C-terminal region of PAP. Applicant has unexpectedly discovered that these mutants are also non-toxic in vivo (i.e., non-phytotoxic) even though they inhibit translation in vitro. Preferred mutants have deletions of from about 26 to about 76 amino acids of mature PAP, and more preferred are the PAP mutants PAP (1-236)-PAP (1-184), inclusive. Thus, truncations beginning at about amino acid residue 237 of wild-type mature PAP, e.g., PAP (1-236), PAP (1-235), PAP (1-234), PAP (1-233), PAP (1-232), PAP (1-231), PAP (1-230), PAP (1-229), PAP (1-228), PAP (1-227), PAP (1-226), PAP (1-225), PAP (1-224), PAP (1-223), PAP (1-222), PAP (1-221), PAP (1-220), PAP (1-219), PAP (1-218), PAP (1-217), PAP (1-216), PAP (1-215), PAP (1-214), PAP (1-213), PAP (1-212), PAP (1-211), PAP (1-210), PAP (1-209), PAP (1-208), PAP (1-207), PAP (1-206), PAP (1-205), PAP (1-204), PAP (1-203), PAP (1-202), PAP (1-201), PAP (1-200), PAP (1-199), PAP (1-198), PAP (1-197), PAP (1-196), PAP (1-195), PAP (1-194), PAP (1-193), PAP (1-192), PAP (1-191), PAP (1-190), PAP (1-189), PAP (1-188), PAP (1-187), PAP (1-186), PAP (1-185), and PAP (1-184) are encompassed by the present invention. More preferred mutants include PAP (1-184Glu), PAP (1-188Lys), PAP (1-206Glu), PAP (1-209) and PAP (1-236Lys). Deletions shorter than about 26 (i.e., between 1 and 25 amino acids, inclusive) or longer than 76 mature PAP amino acids are included in the scope of the present invention provided that they are non-toxic to plant cells, which can be determined by the selection method described in detail below, and they confer fungus and/or virus resistance in planta. The latter properties can be determined in vitro, e.g., by inoculating plant parts, e.g. leaves, with the PAP mutant in the presence of a virus or fungus, or by separate in vivo assays wherein a transgenic plant transformed with a mutant PAP-encoding DNA is inoculated with a fungus or virus. A preferred C-terminal substitution mutant is PAP (1-262, Leu202Phe). Again, while not intending to be bound by any particular theory of operation, Applicant believes that the sequence of PAP amino acids 244Glu-259Cys (shown in Table I), which is homologous to the consensus sequence for the prokaryotic membrane lipoprotein lipid attachment site (Hayashi et al., J. Bioenerg. Biomem. 22:451-471 (1990)), and which is absent from each of the PAP mutants disclosed above, is involved in binding of PAP to phospholipids on endoplasmic reticulum (ER) membranes which facilitates the translocation of PAP into the cytosol of the cell where it inhibits protein synthesis. Disarming this function, e.g., by deletion or by frameshift mutation, results in PAP mutants having the instantly disclosed properties.
Dore also discloses the PAP mutant Phe195Tyr, Lys211Arg (which numbering is +1 out-of-phase with the numbering used herein due to the N-terminal Met residue required for expression in E. coli), which is toxic to eukaryotic cells (such as plants) but non-toxic to procaryotes such as E. coli. Accordingly, this PAP mutant disclosed in the Dore publication is not included within the scope of the present invention.
The third category of PAP mutants is characterized by truncations of from 1 to at least about 38 N-terminal amino acid residues of mature PAP. These mutants include PAP (2-262), PAP (3-262), PAP (4-262), PAP (5-262), PAP (6-262), PAP (7-262), PAP (8-262), PAP (9-262), PAP (10-262), PAP (11-262), PAP (12-262), PAP (13-262), PAP (14-262), PAP (15-262), PAP (16-262), PAP (17-262), PAP (18-262), PAP (19-262), PAP (20-262), PAP (21-262), PAP (22-262), PAP (23-262), PAP (24-262), PAP (25-262), PAP (26-262), PAP (27-262), PAP (28-262), PAP (29-262), PAP (30-262), PAP (31-262), PAP (32-262), PAP (33-262), PAP (34-262), PAP (35-262), PAP (36-262), PAP (37-262), PAP (38-262) and PAP (39-262). Truncations of greater than 38 N-terminal amino acid residues of mature PAP are included within the scope of the present invention to the extent that they exhibit PAP biological activity and reduced phytotoxicity in vivo. These properties may be determined in accordance with the procedures set forth in the working examples, below.
The fourth category of PAP mutants contain active-site mutations which render the PAP molecule enzymatically inactive (as measured by their lack of ability to inhibit translation in vitro and/or in eukaryotic ribosomes). Applicant has surprisingly and unexpectedly found that these mutants exhibit broad spectrum anti-fungal activity when expressed in plants, even though they exhibit negligible PAP anti-viral activity. The putative active site of PAP includes amino acid residues Tyr72, Tyr123, Glu176, Arg179 and Trp208. Accordingly, PAP active site mutants, e.g., which contain a conservative or even non-conservative substitution in the PAP active site or wherein at least one active site amino acid is deleted or replaced by another amino acid, wherein the PAP is rendered enzymatically inactive but retains anti-fungal activity, are encompassed within the present invention. PAP mutants can be tested for enzymatic and anti-fungal activities using the assay procedures described in the working examples, below. A preferred PAP active site mutant is PAP (1-262, Glu176Val).
In regard to the disclosed PAP mutants, the phrase xe2x80x9cwhich differs from wild-type PAP substantially in that . . . xe2x80x9d means that except for the amino acid changes (described above), that are necessary to confer reduced phytotoxicity and anti-viral and/or anti-fungal activity, the amino acid sequences of the mutant PAPs are substantially identical to that of mature PAP. By the term xe2x80x9csubstantially identical,xe2x80x9d it is meant that the PAP mutants of the present invention can be further modified by way of additional substitutions, additions or deletions provided that the resultant PAP mutant retains reduced phytotoxicity and PAP biological activity as defined herein. For example, the N-terminus of the mutant PAP may be changed to a methionine residue, either by substitution or addition, to allow for expression of a DNA encoding the mutant PAP in various host cells particularly E. coli. The PAP mutants of the present invention may further include the N-terminal 22-amino acid signal peptide of wild-type PAP and/or the 29-amino acid C-terminal extension, both of which are shown in Table I above.
DNAs encoding the mutant PAPs of the present invention can be prepared by manipulation of known PAP genes. See Ausubel et al. (eds.), Vol. 1, Chap. 8 in Current Protocols in Molecular Biology, Wiley, N.Y. (1990). The DNAs may also be prepared via PCR techniques. See PCR Protocols, Innis et al. (eds.), Academic Press, San Diego, Calif. (1990). The mutant PAP-encoding DNA (e.g., a cDNA) is preferably inserted into a plant transformation vector in the form of an expression cassette containing all of the necessary elements for transformation of plant cells. The expression cassette typically contains, in proper reading frame, a promoter functional in plant cells, a 5xe2x80x2 non-translated leader sequence, the mutant PAP DNA, and a 3xe2x80x2 non-translated region functional in plants to cause the addition of polyadenylated nucleotides to the 3xe2x80x2 end of the RNA sequence. Promoters functional in plant cells may be obtained from a variety of sources such as plants or plant DNA viruses. The selection of a promoter used in expression cassettes will determine the spatial and temporal expression pattern of the construction in the transgenic plant. Selected promoters may have constitutive activity and these include the CaMV 35S promoter, the actin promoter (McElroy et al., Plant Cell 2:163-171 (1990); McElroy et al., Mol. Gen. Genet. 231:150-160 (1991); Chibbar et al., Plant Cell Rep. 12:506-509 (1993), and the ubiquitin promoter (Binet et al., Plant Science 79:87-94 (1991), Christensen et al., Plant Mol. Biol. 12:619-632 (1989); Taylor et al., Plant Cell Rep. 12:491495 (1993)). Alternatively, they may be wound-induced (Xu et al., Plant Mol. Biol 22:573-588 (1993), Logemann et al., Plant Cell 1:151-158 (1989), Rohrmeier and Lehle, Plant Mol. Bio. 22:783-792 (1993), Firek et al., Plant Mol. Biol. 22:129-142 (1993), Warener et al., Plant J. 3:191-201 (1993)) and thus drive the expression of the mutant PAP gene at the sites of wounding or pathogen infection. Other useful promoters are expressed in specific cell types (such as leaf epidermal cells, meosphyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example). Patent Application WO 93/07278, for example, describes the isolation of the maize trpA gene which is preferentially expressed in pith cells. Hudspeth and Grula, Plant Mol. Biol. 12:579-589 (1989), have described a promoter derived from the maize gene encoding phosphoenolpyruvate carboxylase (PEPC) with directs expression in a leaf-specific manner. Alternatively, the selected promoter may drive expression of the gene under a light-induced or other temporally-regulated promoter. A further alternative is that the selected promoter be chemically regulated. The DNAs may also encode the N-terminal signal sequence and/or the C-terminal extension of wild-type PAP.
A variety of transcriptional cleavage and polyadenylation sites are available for use in expression cassettes. These are responsible for correct processing (formation) of the 3xe2x80x2 end of mRNAs. Appropriate transcriptional cleavage and polyadenylation sites which are known to function in plants include the CaMV 35S cleavage and polyadenylation sites, the tml cleavage and polyadenylations sites, the nopaline synthase cleavage and polyadenylation sites, the pea rbcS E9 cleavage and polyadenylation sites. These can be used in both monocotyledons and dicotyledons.
Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adh1 gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop 1:1183-1200 (1987)). In the same experimental system, the intron from the maize bronze-l gene had a similar effect in enhancing expression (Callis et al., supra.). Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the xe2x80x9cxcexa9-sequencexe2x80x9d), Maize Chlorotic mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al., Nucl. Acids Res. 15:8693-8711 (1987); Skuzeski et al., Plant Mol. Biol. 15:65-79 (1990)).
Numerous transformation vectors are available for plant transformation, and the genes of this invention can be used in conjunction with any such vectors. The selection of vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformations include the nptII gene which confers resistance to kanamycin (Messing and Vierra, Gene 19:259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res. 18:1062 (1990); Spencer et al., Theor. Appl. Genet. 79:625-631 (1990)), the hph gene which confers resistance to the antibiotic hygromycin (Blochinger and Diggelmann, Mol. Cell Biol. 4:2929-2931 (1984)), and the dhfr gene, which confers resistance to methotrexate. Vectors suitable for Agrobacterium transformation typically carry at least one T-DNA border sequence. These include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984) and pCIB200 (EP 0 332 104).
Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques which do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. For example, pCIB3064 is a pUC-derived vector suitable for the direct gene transfer technique in combination with selection by the herbicide basta (or phosphinothricin). It is described in WO 93/07278 and Koziel et al. (Biotechnology 11:194-200 (1993)).
An expression cassette containing the mutant PAP gene DNA containing the various elements described above may be inserted into a plant transformation vector by standard recombinant DNA methods. Alternatively, some or all of the elements of the expression cassette may be present in the vector, and any remaining elements may be added to the vector as necessary.
Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques which do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described in Paszkowski et al., EMBO J 3:2717-2722 (1984), Potrykis et al., Mol. Gen. Genet. 199:169-177 (1985), Reich et al., Biotechnology 4:1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques.
Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. The many crop species which are routinely transformable by Agrobacterium include tobacco, tomato, sunflower, cotton, oilseed rape, potato, soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432 (tomato), WO 87/07299 (Brassica), U.S. Pat. No. 4,795,855 (poplar)). Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident plasmid or chromosomally (e.g. strain CIB542 for pCIB200 (Uknes et al. Plant Cell 5:159-169 (1993)). The transfer of the recombinant binary vector, to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen and Willmitzer, Nucl. Acids Res. 16:9877 (1988)).
Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols known in the art. Transformed tissue is regenerated on selectable medium carrying an antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
Preferred transformation techniques for monocots include direct gene transfer into protoplasts using PEG or electroporation techniques and particle bombardment into callus tissue. Transformation can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with this invention. Co-transformation may have the advantages of avoiding complex vector construction and generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al., Biotechnology 4:1093-1096 (1986)).
Published European Patent Applications EP 0 292 435 and EP 0 392 225, and PCT application WO 93/07278 describe techniques for the preparation of callus and protoplasts of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordeon-Kamm et al., Plant Cell 2:603-618 (1990), and Fromm et al., Biotechnology 11:194-200 (1993), describe techniques for the transformation of elite inbred lines of maize by particle bombardment.
Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-types (Zhange et al., Plant Cell Rep. 7:739-384 (1988); Shimamoto et al., Nature 338:274-277 (1989); Datta et al., Biotechnology 8:736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al., Biotechnology 9:957-962 (1991)).
Patent Application EP 0 332 581 described techniques for the generation, transformation and regeneration of Pooideae protoplasts. Furthermore wheat transformation has been described by Vasil et al. (Biotechnology 10:667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology 11:1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102:1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus.
Transformation of monocot cells such as Zea mays can be achieved by bringing the monocot cells into contact with a multiplicity of needle-like bodies on which these cells may be impaled, causing a rupture in the cell wall thereby allowing entry of transforming DNA into the cells. See U.S. Pat. No. 5,302,523. Transformation techniques applicable to both monocots and dicots are also disclosed in the following U.S. Pat. No. 5,240,855 (particle gun); U.S. Pat. No. 5,204,253 (cold gas shock accelerated microprojectiles); U.S. Pat. No. 5,179,022 (biolistic apparatus); U.S. Pat. Nos. 4,743,548 and 5,114,854 (microinjection); and U.S. Pat. Nos. 5,149,655 5,120,657 (accelerated particle mediated transformation); U.S. Pat. No. 5,066,587 (gas driven microprojectile accelerator); U.S. Pat. No. 5,015,580 (particle-mediated transformation of soy bean plants); U.S. Pat. No. 5,013,660 (laser beam-mediated transformation); and U.S. Pat. Nos. 4,849,355 and 4,663,292.
The thus-transformed plant cells or plant tissue are then grown into full plants in accordance with standard techniques. Transgenic seed can be obtained from transgenic flowering plants in accordance with standard techniques. Likewise, non-flowering plants such as potato and sugar beets can be propagated by a variety of known procedures. See, e.g., Newell et al., Plant Cell Rep. 10:30-34 (1991) (disclosing potato transformation by stem culture).
The mutant PAP encoding DNAs of the present invention confer broad spectrum fungus and virus resistance to any plant capable of expressing the DNAs, including monocots (e.g., cereal crops) and dicots. Specific examples include maize, tomato, turfgrass, asparagus, papaya, sunflower, rye, beans, ginger, lotus, bamboo, potato, rice, peanut, barley, malt, wheat, alfalfa, soybean, oat, eggplant, squash, onion, broccoli, sugarcane, sugar beet, beets, apples, oranges, grapefruit, pear, plum, peach, pineapple, grape, rose, carnation, daisy, tulip, Douglas fir, cedar, white pine, scotch pine, spruce, peas, cotton, flax and coffee.
PAP mutants other than those specifically described above can be identified by a selection system in eukaryotic cells. In a preferred embodiment, a PAP-encoding DNA molecule, operably linked to an inducible promoter functional in the eukaryotic cell, is randomly mutagenized in accordance with standard techniques. The cell is then transformed with the mutagenized PAP construct. The thus-transformed cell is then cultured in a suitable medium for a predetermined amount of time, e.g., sufficient to cause some growth of the cells, at which time an inducer is added to the medium to cause expression of the mutagenized DNA molecule. If the cultured cell survives the induction of the expression of the mutagenized PAP DNA molecule, which indicates that the mutagenesis resulted in the expression of a non-toxic PAP mutant, the PAP mutant can be then assayed in vitro or in vivo to determine whether it retains PAP biological activity. Preferred in vitro assays include eukaryotic translation systems such as reticulocyte lysate systems wherein the extent of the inhibition of protein synthesis in the system caused by the PAP mutant is determined. Preferred host cells are yeast cells such as Saccharomyces cerevisiae, as described in greater detail in Example 1, below. This method can also be conducted with a plurality of randomly mutagenized PAP-encoding DNA molecules. The PAP mutants identified as having reduced phytotoxicity and which retain PAP anti-viral and/or anti-fungal activity, as determined by subsequent assays, can then be isolated, purified and sequenced in accordance with standard techniques.
In another embodiment, the mutagenesis is performed after the transformation of the eukaryotic cell. The disadvantage with mutagenizing the DNA after transformation is that the chromosomal DNA of the host can also be mutagenized. To determine whether the mutations of the surviving cells are chromosomal or plasmid-borne in nature, this embodiment requires the step of replacing the transforming PAP-encoding DNA with wild-type PAP-encoding DNA under the control of an inducible promoter, and growing the cells in the presence of the inducer. Mutants which retain the ability to grow are chromosomal mutants, whereas mutants which fail to grow are plasmid-borne (i.e., PAP) mutants.